Fig. 3. Periodic CB₁ hotspots display stable dynamics revealed by live SIM imaging.

a Representative live image of transfected CB₁-RFP in the primary neuron (DIV 9–12) of the SD rat acquired by SIM. Individual CB₁ hotspots are marked with purple balls, and locations of each individual time points are connected with lines. Line colors indicate trace indexes. N = 3 biological replicates. b Five CB₁ hotspots shown in (a) with their relative locations. c Displacement changes from (b) are around 60–70 nm between neighboring time points. d Averaged autocorrelation analysis of CB₁ distributions at different time points with the histogram of the autocorrelation amplitude. p = 0.99 (no significance), one-way ANOVA. Actual autocorrelation from left to right, 0.47 ± 0.02, 0.47 ± 0.03, 0.47 ± 0.02. e Averaged cross-correlation analysis between the neighboring frames (0 vs 60 s, 0 vs 120 s) showed similar distribution properties and histogram showing amplitude of average cross-correlation. p = 0.386 (no significance), statistical analysis was performed by unpaired two-tailed Student’s t test. Actual cross-correlation amplitude (from left to right), 0.45 ± 0.05; 0.51 ± 0.02. f The histogram of CB₁ spacing across time points. p = 0.69 (no significance, one-way ANOVA). Actual spacing (from left to right), 192 ± 0.6, 192 ± 0.7, 191 ± 0.6 nm. Data in (d−f) are mean ± s.e.m. (N = 3 biological replicates; 70–120 axonal regions were examined per condition). g Traces of the individual CB₁ hotspots over time. Source data are provided as a Source Data file.